1. Mark W. Luckenbach 1, Elizabeth North 2, M. Lisa Kellogg 3, Roger Mann 4, Steve M. Allen 5 and Kennedy T. Paynter 2,3 Fertilization Success in Altered Bivalve Populations: Implications for Restoration 1 Virginia Institute of Marine Science, College of William and Mary, Eastern Shore Laboratory 2 University of Maryland Center for Environmental Studies, 3 University of Maryland 4 Virginia Institute of Marine Science, College of William and Mary 5 Oyster Recovery Partnership

2. Problem Description Fertilization success in free-spawning, sessile marine bivalves is dependent upon gametes making contact. Many of the shellfish populations that we seek to protect or restore currently exist at low population density, e.g., Argopecten irradians Mercenaria mercenaria Crassostrea virginica Arctica islandica Restoration activities that add bivalves as broodstock do so with little basis for determining the density required to achieve high fertilization success. Moreover, the addition of broodstock is often done without regard for the sex ratio, anticipated gamete production or potential fertilization success.

3. Problem Description High fecundity in these species can lead to the expectation that a small population size is sufficient to effect a recovery. For instance: 10 6 -10 7 eggs per at a density of 10 0 – 10 2 s m -2 yields 10 10 – 10 13 eggs hectare -1 Assumptions about how many of these eggs are fertilized has the potential to propagate errors through demographics models that are several orders of magnitude.

4. High Sperm:Egg Bolingbroke Sand Low Sperm:Egg Potentially Sperm Limited Some Preliminary Findings with Crassostrea virginica (Kellogg et al. 2007 ICSR) 10 8 10 9 10 10 11 10 12

5. Objectives 1) Better understand the factors that affect fertilization success, including… gamete concentrations fertilization efficiency (avg. # sperm to fertilize an egg) turbulent mixing 2) Ultimately, we want to use this, coupled with density, size and fecundity estimates from the field, to estimate not only egg production, but also fertilization success in natural and restored bivalve populations.

6. Approach 1)Construct and parameterize a computational model which predicts fertilization success based upon contact between gametes in a turbulent medium. 2) Conduct laboratory experiments with Crassostrea gametes using field-relevant turbulent mixing conditions to test initial model predictions. 3)Refine the model predictions using experimental results. 4) Repeat 2 – 3 as necessary.

7. Predicts concentration of fertilized eggs over time when mixed with sperm of a given concentration: where U = concentration of unfertilized eggs (number cm -3 ) and F = concentration of fertilized eggs (number cm -3 ) f = fertilization constant (=1 if every contact results in fertilization) dt = time interval (s) ε = contact rate of sperm with each egg (number s -1 ). Fertilization Success Model

8. The contract rate of egg and sperm (E) [After Rothschild & Osborn (1988), Evans (1989), Visser & MacKenzie (1998)] : where c = concentration of prey particles (e.g., sperm concentration, number cm -3 ) and R = reactive distance of predator particles (e.g., effective egg radius, cm) u = swimming velocity of prey particles (e.g., sperm swimming speed, cm s -1 ) v = randomly directed motion of predator particles (e.g., v = 0 for eggs) w = root-mean-square turbulent velocity, (cm s -1 ): where a = constant [1.37 or 1.9 according to references in Visser and MacKenzie (1989)] d = turbulence length scale [d = R according to Visser and MacKenzie (1989)] ε = turbulent dissipation rate (cm 2 s -3 ).

9. Unfertilized egg conc. = 100 cm -3 Sperm concentration = 1000 cm -3 Fertilization efficiency = 0.01 Unfertilized egg conc. = 100 cm -3 Sperm concentration = 1000 cm -3 Fertilization efficiency = 0.03 Unfertilized egg conc. = 100 cm -3 Sperm concentration = 1000 cm -3 Fertilization efficiency = 0.1 Unfertilized egg conc. = 100 cm -3 Sperm concentration = 1000 cm -3 Fertilization efficiency = 0.2 Unfertilized egg conc. = 100 cm -3 Sperm concentration = 1000 cm -3 Fertilization efficiency = 0.5 Unfertilized egg conc. = 100 cm -3 Sperm concentration = 1000 cm -3 Fertilization efficiency = 1.0 Preliminary model run examples Turbulent energy dissipation rates ε = 0.007 cm 2 s -3 ε = 0.034 cm 2 s -3 ε = 0.180 cm 2 s -3

10. Solutions for contact rates when the following parameters were varied: A) sperm concentration (note the log scale), B) sperm swimming speed and contact (egg) radius, and C) turbulent dissipation rate (e) and the constant a. Open symbols indicate base-case solutions.

11. Turbulent mixing Examples of initial release of dye (left) and final distribution of dye (right) in grid chambers. Turbulent energy dissipation rates 1) measured in Chesapeake Bay and 2) occurring in the grid-stirred chambers with different motor speeds (colored lines). Field data courtesy of Larry Sanford, UMCES Horn Point Laboratory Average turbulent energy dissipation rates (e, cm 2 s -3 ), average turbulent shear (g, s -1 ), and Reynolds number at different motor speeds (RPM) predicted for the grid- stirred chambers. Grid-stirred chambers Mixing controlled by motor speed

12. Summary of sperm dilution experiments with species, gamete concentrations, gamete ratios and mean % of eggs fertilized. Values for % fertilized are means of three treatment replicates. Species Sperm Dilution Gamete Concentration Sperm ml -1 Eggs ml -1 Sperm:Egg Mean % Fertilized 10 1 9.63 x 10 6 10 4 9.63 x10 2 66.67 10 2 9.63 x 10 5 10 4 9.63 x10 1 8.94 C. virginica 10 3 9.63 x 10 4 10 4 9.63 x10 0 2.06 10 4 9.63 x 10 3 10 4 9.63 x10 -1 2.22 10 5 9.63 x 10 2 10 4 9.63 x10 -2 1.89 10 6 9.63 x 10 1 10 4 9.63 x10 -3 2.28 10 7 9.63 x 10 0 10 4 9.63 x10 -4 1.94 10 1 1.71 x 10 7 10 4 1.71 x 10 3 93.67 10 2 1.71 x 10 6 10 4 1.71 x 10 2 69.83 C. ariakensis 10 3 1.71 x 10 5 10 4 1.71 x 10 1 9.00 10 4 1.71 x 10 4 10 4 1.71 x 10 0 0.33 10 5 1.71 x 10 3 10 4 1.71 x 10 -1 0.00 10 6 1.71 x 10 2 10 4 1.71 x 10 -2 0.00 10 7 1.71 x 10 1 10 4 1.71 x 10 -3 0.00 10 0 1.46 x 10 6 10 3 1.46 x 10 3 92 C. ariakensis 10 2 1.46 x 10 4 10 3 1.46 x 10 1 12.5 10 4 1.46 x 10 2 10 3 1.46 x 10 -1 0 C. ariakensis 10 0 9.19 x 10 5 10 2 9.19 x 10 3 99.25 10 2 9.19 x 10 3 10 2 9.19 x 10 1 81.63 10 4 9.19 x 10 1 10 2 9.19 x 10 -1 1.25 C. virginica 10 1 9.16 x 10 5 10 2 9.16 x 10 3 97.5 10 3 9.16 x 10 3 10 2 9.16 x 10 1 53.5 10 5 9.16 x 10 1 10 2 9.16 x 10 -1 0.33 C. virginica 10 1 3.43 x 10 5 10 3 3.43 x 10 2 55.67 10 3 3.43 x 10 3 10 3 3.43 x 10 0 13.50 10 5 3.43 x 10 1 10 3 3.43 x 10 -2 0.50 C. virginica 10 1 1.42 x 10 6 10 1 1.42 x10 5 90 10 3 1.42 x 10 4 10 1 1.42 x10 3 94 10 5 1.42 x 10 2 10 1 1.42 x10 1 66.17

13. Summary of sperm dilution experiments with species, gamete concentrations, gamete ratios and mean % of eggs fertilized. Values for % fertilized are means of three treatment replicate. Species Egg Dilution Gamete Concentration Sperm:Egg % Fertilized Sperm ml -1 Eggs ml -1 C. virginica 10 3 5.54 x 10 4 10 1 5.54 x 10 3 98.50 10 2 5.54 x 10 4 10 2 5.54 x 10 2 88.17 10 1 5.54 x 10 4 10 3 5.54 x 10 1 35.67 C. virginica 10 3 6.98 x 10 4 10 1 6.98 x 10 3 97.17 10 2 6.98 x 10 4 10 2 6.98 x 10 2 97.17 10 1 6.98 x 10 4 10 3 6.98 x 10 1 67.67 C. virginica 10 3 6.98 x 10 4 10 1 6.98 x 10 3 94.17 10 2 6.98 x 10 4 10 2 6.98 x 10 2 65.83 10 1 6.98 x 10 4 10 3 6.98 x 10 1 33.67

14. Examples of mean % eggs fertilized at varying sperm density C. virginica 100 eggs / ml C. ariakensis 100 eggs / ml C. virginica 10,000 eggs / ml C. ariakensis 10,000 eggs / ml

15. 10 6 10 5 10 4 10 3 10 2 10 1 10 0 10 -1 10 -2 10 -3 10 -4 Percent fertilization as a function of sperm : egg ratios where x was the ratio of initial sperm and unfertilized egg conc. Fertilization Efficiency =

16. Model predictions of fertilization success after 30 min at different initial egg concentrations: A) 10 eggs cm 3, B) 100 eggs cm 3, and C) 1000 eggs cm 3. (1 RPM, ε = 0.00067 cm 2 s -3 ; 3 RPM, ε = 0.007 cm 2 s -3 ; 6 RPM, ε = 0.034 cm 2 s -3 ; 12 RPM, ε = 0.18 cm 2 s -3 ).

17. Conclusions Observed fertilization success is very low at sperm:egg ratios 10 2 and generally only high above 10 3...... even at high gamete concentrations. Model predictions suggest that at high mixing rates fertilization success below this sperm:egg threshold should increase...... however, this neglects the high dilution rate at high turbulence.

18. Implications Under conditions of low population density, such as exists for many overexploited bivalve species, or when a single age class dominates the population, as often exists in areas of sporadic recruitment or at restoration sites where single cohorts are planted, population growth may be seriously constrained by low fertilization success.

19. Future Directions Investigate fertilization success in the laboratory at higher mixing rates. Make the model open, i.e. allow for dilution of gametes. Extend the experiments to other bivalve species. Conduct fine scale spatial surveys of restored bivalve populations to more accurately map densities and distributions of the sexes.

20. Acknowledgments Funding was provided by the NOAA Chesapeake Bay Office and the Keith Campbell Foundation for the Environment. We thank Larry Sanford and Steve Suttles for field data and construction of the turbulence mixing chamber, respectively.

21. Sperm swimming speed Sperm and egg suspensions were added to opposite ends of a flat capillary tube. Observed under an inverted light microscope with attached low light video camera. With a frame grab frequency of 0.01 s -1 we measured the movement of individual sperm for 0.6 – 1.0 s. 8 cm 3.5 mm Egg suspensionSperm suspension Number Swimming speed (mm/sec) Measured distribution of swimming speeds for C. virginica sperm. Each bar is from a single video record and observations are ranked in increasing order. Mean υ = 0.0057 cm s -1

23. Species, sample size and range of shell heights used in determination of male size-specific fecundity. Species n Range in Shell Height (mm) Crassostrea virginica5517.4 – 135.7 C. ariakensis (West Coast strain)1865.3 – 140.1 C. ariakensis (South China strain)2362.1 – 88.0 0 100 200 300 400 050100150 Shell Height (mm ) Estimated sperm (in billions) vs. shell height for Crassostrea virginica ( ), west coast strain of C. ariakensis ( ) and south China strain of C. ariakensis ( ). Size-specific Fecundity in Males